The Bramwell-Hill model describes the relation between vascular wall stiffness expressed in aortic distensibility and the pulse wave velocity (PWV), which is the propagation speed of the systolic pressure wave through the aorta. The main objective of this study was to test the validity of this model locally in the aorta by using PWV-assessments based on in-plane velocity-encoded cardiovascular magnetic resonance (CMR), with invasive pressure measurements serving as the gold standard.Seventeen patients  awaiting cardiac catheterization were prospectively included. During catheterization, intra-arterial pressure measurements were obtained in the aorta at multiple locations 5.8 cm apart. PWV was determined regionally over the aortic arch and locally in the proximal descending aorta. Subsequently, patients underwent a CMR examination to measure aortic PWV and aortic distention. Distensibility was determined locally from the aortic distension at the proximal descending aorta and the pulse pressure measured invasively during catheterization and non-invasively from brachial cuff-assessment. PWV was determined regionally in the aortic arch using through-plane and in-plane velocity-encoded CMR, and locally at the proximal descending aorta using in-plane velocity-encoded CMR. Validity of the Bramwell-Hill model was tested by evaluating associations between distensibility and PWV. Also, theoretical PWV was calculated from distensibility measurements and compared with pressure-assessed PWV.In-plane velocity-encoded CMR provides stronger correlation (p = 0.02) between CMR and pressure-assessed PWV than through-plane velocity-encoded CMR (r = 0.69 versus r = 0.26), with a non-significant mean error of 0.2 ± 1.6 m/s for in-plane versus a significant (p = 0.006) error of 1.3 ± 1.7 m/s for through-plane velocity-encoded CMR. The Bramwell-Hill model shows a significantly (p = 0.01) stronger association between distensibility and PWV for local assessment (r = 0.8) than for regional assessment (r = 0.7), both for CMR and for pressure-assessed PWV. Theoretical PWV is strongly correlated (r = 0.8) with pressure-assessed PWV, with a statistically significant (p = 0.04) mean underestimation of 0.6 ± 1.1 m/s. This theoretical PWV-estimation is more accurate when invasively-assessed pulse pressure is used instead of brachial cuff-assessment (p = 0.03).CMR with in-plane velocity-encoding is the optimal approach for studying Bramwell-Hill associations between local PWV and aortic distensibility. This approach enables non-invasive estimation of local pulse pressure and distensibility. The pathophysiological processes of cardiovascular disease involve stiffening of the arterial vessel wall. Increased aortic wall stiffness results in an increased aortic pulse pressure and left ventricular afterload, restricting left ventricular filling during diastole, which eventually may lead to heart failure ,2. AdditAssessment of regional PWV is of high interest in cardiovascular research as the independent prognostic value of regional PWV-assessment for outcome prediction in various patient populations has been recognized -4,7-10. Several estimators - such as Young's modulus, distensibility or stiffness index - are currently in use to express aortic stiffness, all relating local blood pressure with the distention of the aorta . Aortic distensibility is defined as the relative change in vessel diameter over local pulse pressure ,14. A usThe gold standard for PWV-assessment is defined from invasive pressure measurements at consecutive locations in the aorta, from which the propagation speed of the systolic pressure wave front can be accurately determined. Cardiovascular Magnetic Resonance (CMR) with velocity-encoding (VE) is a validated alternative for measuring the PWV , globallThe Bramwell-Hill model  theoretiData from the subjects reported in this study have been described earlier in studies validating PWV-assessment with VE-CMR ,21. ResePatients underwent CMR examination to measure aortic PWV and aortic distention. The mean interval between catheterization and CMR was 17 ± 13 days. Exclusion criteria were general contraindications to CMR, evidence of aortic valve stenosis on ultrasonography, coarctation of the aorta, or other congenital heart disease, or a family history of Marfan syndrome.x) by a wave (pressure or blood flow) divided by the transit-time (Δt) for the wave to travel that distance: PWV = Δx/Δt. This definition holds true under the assumption that no wave reflections occur, as the transmission of the pressure pulse as a sum of incident and reflected waves does not represent the true PWV [PWV is defined as the distance traveled (Δtrue PWV . Alternatrue PWV . The Bratrue PWV  as follo-3) and Distensibility defined by dV/(V∙dP), with V the aortic volume and P the blood pressure. Distensibility can be estimated by the relative luminal area change over the local pulse pressure:with ρ being the blood density  and VE CMR, with blood pressure assessment both invasively and from brachial cuff measurement, and assessment of aortic luminal area distention with CMR.pressure is calculated as Δx/Δt (expressed in m/s), where Δx is the aortic path length between measurement sites, determined from catheter pullback, and Δt is the transit-time for the systolic pressure wave front to propagate between these sites. The onset of the systolic pressure wave front was automatically determined from the time point (relative to the R-wave) of minimal pressure prior to the upslope of the systolic pressure wave. Local pulse pressure was determined from the difference between maximal and minimal pressure. Offline analysis of the pressure-time curves was performed using custom-made software.Invasive pressure-time curves and simultaneous ECG recordings were obtained immediately after vascular access, to avoid any interference by medication or performed procedures. A 6F JR4 pressure tip catheter  was introduced through a 6F sheet (Cordis) into either one of the femoral arteries and advanced through the aorta until just distal to the aortic valve. During stepwise pullback, pressure waves were recorded at consecutive positions spaced 5.8 cm apart. Pressure-time curves and ECGs were recorded with a sampling resolution of 2 kHz during at least 10 cardiac cycles at each position to take into account variations induced by respiration. PWVt.p.), planned perpendicular to the aorta and transecting both the ascending aorta , both regionally for the same aortic arch trajectory as well as locally in the aorta at the imaging location of site 2.VE CMR was performed on a 1.5 Tesla CMR scanner with a typical total acquisition time of 25 minutes . PWV was assessed regionally for the aortic arch using a single one-directional through-plane VE acquisition  direction and in frequency-encoding  direction respectively with velocity sensitivity set to 150 cm/s in either direction. The body coil was used for signal reception. Scan parameters were: 60%-rectangular FOV 450 × 270 mm2, 10 mm slice thickness, TE 2.4 ms, TR 4.3 ms, α 10°, acquisition voxel size 3.5 × 2.1 × 10.0 mm3, sampling bandwidth 495 Hz, NSA 2, retrospective-gating with maximal number of phases reconstructed into one average cardiac cycle with arrhythmia rejection window set to acceptance of 15%-variation. Acquisition was performed with free breathing without respiratory compensation and scan time of a single acquisition amounted to 7 minutes 42 seconds at a heart rate of 60 bpm.Finally, PWVi.p. was obtained as described before [For CMR-assessed PWV, Δx/Δt was calculated with Δx the aortic path length between measurement sites and Δt the transit-time for the systolic velocity wave front to propagate between these sites. The aortic path length between the subsequent measurement sites in ascending and proximal descending aorta was manually assessed by drawing a poly-line along the centerline of the aortic arch within the scout images, using the in-house developed software package MASS  . At eachd before , from tht.p. and PWVi.p. were determined regionally in the aortic arch between measurement sites 1 and 2 and PWVi.p. was determined also locally around site 2, for an 11.6 cm trajectory starting 5.8 cm above site 2 to 5.8 cm below site 2. The aortic luminal distension was determined at site 2 from the maximal and minimal cross-sectional lumen area measured as described above with the automated contour detection on the velocity-insensitive magnitude gradient-echo images. Immediately after VE CMR-examination, brachial cuff blood pressure was obtained using a semi-automated sphygmomanometer , with the subject remaining in supine position on the CMR-table. Regional PWVpressure was determined over the aortic arch, from pressure measurements between sample points closest to measurement sites 1 and 2. Furthermore, local PWVpressure was determined for an 11.6 cm trajectory around site 2.PWVStatistical analysis was performed using SPSS for Windows . All data are presented as mean values ± one SD, unless stated otherwise.t.p. and PWVi.p. with PWVpressure were evaluated using the Pearson's correlation coefficient (r), while variation with PWVpressure was studied with coefficients of variation . Also mean unsigned error (with PWVpressure as reference standard) and 95%-confidence intervals (95%-CI) were calculated. Statistical significance of differences between correlation coefficients was tested by stepwise linear regression analysis with the gold standard and the interaction between the gold standard and the tested methods as predictors. The approach described by Bland and Altman [cuff) and intra-arterially during catheterization (i.e. PPcath). Furthermore, the estimated theoretical PWV from distensibility assessment was compared to the gold standard PWVpressure. Statistical significance on all tests was indicated by p < 0.05.Associations of PWVd Altman  was follPatient characteristics are presented in Table Regionally in the aortic arch, mean PWV from in-plane VE CMR was 6.7 ± 2.3 m/s, mean PWV from through-plane was 5.6 ± 1.2 m/s and mean PWV from invasive pressure measurements was 6.9 ± 1.5 m/s. Locally at site 2, mean PWV from in-plane VE CMR was 7.1 ± 2.6 m/s and from invasive pressure measurement 7.0 ± 1.6 m/s.t.p. and PWVi.p. regionally for the aortic arch and PWVi.p. locally at the proximal descending aorta were compared against PWVpressure. In Figure pressure was high . Mean unsigned error for PWVt.p. was 26% and for PWVi.p. 18%. For local PWV-assessment, correlation was strong  and variation amounted to 19%. The mean unsigned error was 15%. Only regional PWVt.p. was significantly different from PWVpressure . From the Bland-Altman plot, it is clear that this underestimation increases with increasing PWVpressure.First, PWVt.p., PWVi.p. and PWVpressure regionally in the aortic arch and PWVi.p. and PWVpressure locally at the proximal descending aorta) with -1/2  are presented in Table i.p. with distensibility from PPcuff and r = 0.83, p < 0.001 for PWVpressure with distensibility from PPcath) with (distensibility)-1/2 than regional PWV-assessment . Furthermore, PWVpressure showed strong correlation  which was comparable (p ≥ 0.24) with CMR-assessed PWV . None of the associations were significantly different when using PP-values from brachial cuff assessment or intra-arterially during catheterization.Next, the validity of the Bramwell-Hill model was tested. Associations between all PWV-assessments (i.e. PWVcuff and PPcath). Correlations between these modeled local PWV-values and the gold standard  are presented in Figure pressure was strong  and comparable (p = 0.27) for pressure measurements with brachial cuff and during catheterization, with COV of 14% for PPcuff and 17% for PPcath and mean unsigned errors of 16% for both PPcuff and PPcath. The Bland-Altman plot also shows a significant higher (p = 0.03) underestimation of the modeled PWV-values for PPcuff (mean underestimation 1.1 ± 0.9 m/s (p < 0.001)) than for PPcath (mean underestimation 0.6 ± 1.1 m/s (p = 0.04)).Finally, modeled local PWV-values were calculated from the measured local distensibility at the proximal descending aorta , both locally at the proximal descending aorta and regionally in the aortic arch. The main findings of our study are: 1) correlation between the gold standard and regional PWV-assessment in the aortic arch with in-plane VE CMR is significantly stronger than with through-plane VE CMR; 2) local PWV-assessment in the proximal descending aorta with in-plane VE CMR is stronger correlated with the gold standard than regional PWV-assessment in the aortic arch; 3) the Bramwell-Hill model shows stronger association between PWV, distensibility and pulse pressure when applied to local PWV-assessment as compared to regional assessment; 4) local PWV modeled from local distensibility is significantly correlated with the gold standard, but shows a significant underestimation, with a higher mean error for pulse pressure measurement from non-invasive brachial cuff than from invasive pressure assessment.Many pathophysiological processes of cardiovascular diseases involve increased stiffening of the arterial vessel wall ,2, whichIn a previous study by Westenberg et al. it was already shown that PWV-assessment from in-plane VE CMR results in a more accurate estimation for global PWV in the complete aorta . Our finIn this study, the validity of the Bramwell-Hill model was evaluated locally at the proximal descending aorta. This sampling location is technically attractive and pathophysiologically relevant. Motion blurring due to breathing and through-plane motion due to cardiac contraction in the ascending aorta may affect aortic lumen measurements, which are required for distensibility calculations. Respiratory motion will have a similar effect on the lumen assessment both in systole as in diastole. At the location of the descending aorta, the aorta will be subjected to minimal through-plane motion. Furthermore, it is known that the aorta stiffens with age. It was shown that global PWV increases in a nonlinear fashion with age and is most pronounced in the thoracic aorta , probablTo our knowledge, we are the first to test the validity of the Bramwell-Hill model applied at a single location in the aorta. We found a stronger association between PWV, distensibility and pulse pressure for local assessment than for regional assessment. Dogui et al. tested the model on a regional level in the aortic arch and acknowledged the inability to assess local values as a limitation to their study . NeverthThe Bramwell-Hill model can be applied to calculate local PWV-values from distensibility measurements, or vice versa. Theoretically, local pulse pressure can be determined non-invasively from this model, by using local PWV- and aortic luminal distention assessments. Still, in our study a significant underestimation for calculated PWV-values were found when compared to PWV-measurements assessed with invasive pressure. However, this mean underestimation amounted to 0.6 m/s, which may not be clinically relevant since this is in order of 10% of normal values for aortic PWV. Several sources of error might explain this underestimation. First, the CMR and catheterization were not performed on the same day, which is a limitation of our study. The time interval between both examinations might be a possible explanation for the differences, since arterial compliance is subject to physiological day-to-day differences in blood pressure, blood flow and sympathic tone . Also, iOur study has some additional limitations. Automated cuff blood pressure measurements were performed for brachial blood pressure assessment immediately after CMR. The gold standard for measuring brachial blood pressure noninvasively is the mercury manometer. In our study a Dinamap automated blood pressure measurement device was used. It has been described that automated sphygmomanometers are less accurate than mercury manometers. Beaubien et al. showed that only 59% of systolic and 56% of diastolic readings will be within 5 mmHg of the mercury manometer values . Due to Finally, the small sample size is another limitation. This study described a validation of the Bramwell-Hill model for local and regional aortic PWV by VE CMR. No data on the prognostic value of PWV-assessment were presented. Large scale studies, both in patients and healthy volunteers, are needed for further testing the validity of the Bramwell-Hill model and to assess whether local PWV from in-plane VE CMR is superior to regional PWV-assessment for predicting outcome. Additionally, PWV-assessment from in-plane VE CMR has been proven to be accurate and reproducible , but imaCMR with in-plane velocity-encoding is the optimal approach for studying the association between local PWV and aortic distensibility as described by the Bramwell-Hill model. This approach enables local pulse pressure and local distensibility estimation in a non-invasive manner.The authors declare that they have no competing interests.JW is responsible for conception and design of this study, data acquisition, analysis and interpretation of the results and drafting of the manuscript; EvP carried out data analysis, interpretation of the results and drafting of the manuscript; HG carried out data acquisition and revising of the manuscript; PS carried out data acquisition, data analysis, interpretation of the results and revising of the manuscript; WJ carried out data acquisition, interpretation of the results and revising of the manuscript; AdR is responsible for conception and design of this study, interpretation of the results and drafting of the manuscript. All authors read and approved the final manuscript.


Nanobubbles (<200 nm in diameter) have several unique properties such as long lifetime in liquid owing to its negatively charged surface, and its high gas solubility into the liquid owing to its high internal pressure. They are used in variety of fields including diagnostic aids and drug delivery, while there are no reports assessing their effects on the growth of lives. Nanobubbles of air or oxygen gas were generated using a nanobubble aerator . Brassica campestris were cultured hydroponically for 4 weeks within air-nanobubble water or within normal water. Sweetfish (for 3 weeks) and rainbow trout (for 6 weeks) were kept either within air-nanobubble water or within normal water. Finally, 5 week-old male DBA1/J mice were bred with normal free-chaw and free-drinking either of oxygen-nanobubble water or of normal water for 12 weeks. Oxygen-nanobubble significantly increased the dissolved oxygen concentration of water as well as concentration/size of nanobubbles which were relatively stable for 70 days. Air-nanobubble water significantly promoted the height , length of leaves , and aerial fresh weight  of Brassica campestris compared to normal water. Total weight of sweetfish increased from 3.0 to 6.4 kg in normal water, whereas it increased from 3.0 to 10.2 kg in air-nanobubble water. In addition, total weight of rainbow trout increased from 50.0 to 129.5 kg in normal water, whereas it increased from 50.0 to 148.0 kg in air-nanobubble water. Free oral intake of oxygen-nanobubble water significantly promoted the weight  and the length  of mice compared to that of normal water. We have demonstrated for the first time that oxygen and air-nanobubble water may be potentially effective tools for the growth of lives. Then nanobubbles were generated using a nanobubble aerator , gas-liquid mixing system with hydrodynamic function . In thisDissolved oxygen concentration (DO) of oxygen-nanobubble water during generation by BUVITAS was measured sequentially by Winkler's method Brassica campestris (32 stumps in each group) were cultured hydroponically for 4 weeks within air-nanobubble water or within normal water. Water was kept circulated (38 L/min) in the tank (125 L) and air-nanobubble solution was continuously generated using the nanobubble aerator. The water level of the tank and water temperature , pH, and other growth-affecting elements  of cultured water were also monitored sequentially. To assess the effect of air-nanobubbles on the growth of fishes, sweetfish (for 3 weeks) and rainbow trout (for 6 weeks) were kept either within air-nanobubble water  or within normal water. Normal fishbait was provided by 13% of primary total weight per day in each group. Finally, to assess the effect of oxygen-nanobubbles on the growth of mice, 5 week-old male DBA1/J mice were bred for 12 weeks with free-normal chaw  and free-drinking either of oxygen-nanobubble distilled water or of normal distilled water. All distilled water was purchased from Otsuka Pharmaceutical Factory, Inc. . Oxygen-nanobubbles distilled water was filtered immediately after generation, and normal distilled water was filtered immediately after opening the lid, with 0.22 um pore size cellulose acetate membrane  to avoid contamination. After filtration, DO was measured by handheld dissolved oxygen meter DO-24 P . Size  and concentration of nanoparticles were measured by NanoSight LM10-HSBT14 . Water was kept in 4°C for 12 weeks and sufficiently supplied in water bottles of mice twice a week after restoring to room temperature. All mice were purchased from CLEA Japan , and housed in a room under controlled temperature (23±1°C) and humidity (45–65%).Data are expressed as mean ± standard error (SE) in the growth of Brassica campestris. Other data are expressed as mean ± standard deviation (SD). Differences between groups were assessed by Student's t-test. Any P value<0.05 was considered statistically significant.Oxygen concentration was 7.7 mg/L in original normal distilled water, whereas it increased to 31.7 mg/L in oxygen-nanobubble water immediately after running nanobubble aerator with 100 L water for 30 min . Figure Air-nanobubble water significantly promoted the height , length of leaves , and aerial fresh weight  of Brassica campestris compared to normal water after hydroponical culture for 4 weeks . DO of cAfter 3 weeks, total weight of sweetfish increased from 3.0 to 6.4 kg in normal water, whereas it increased from 3.0 to 10.2 kg in air-nanobubble water . In addiIn our preliminary experiments, hyperoxidation as much as about 31.7 mg/L of DO in oxygen-nanobubble water took place immediately after generation , but it After 12 weeks, free oral intake of oxygen-nanobubble distilled water significantly promoted the weight  and the length  of mice as compared to free oral intake of normal water  A, C, D.2) values in water to greater extent than that of microbubbles To the best of our knowledge, we demonstrated for the first time that air and oxygen-nanobubbles promote the growth of plants, fishes, and mice. Yoshida et al. demonstrated that cucumber plants at higher dissolved oxygen concentration showed increased area and weight of leaves In this study, there were no obvious abnormalities or harmful effects in fishes and mice with oral intake of air and oxygen-nanobubbles, respectively. No morphological irregularity was recongnized in Brassica campestris cultured with air-nanobubble water, either. It has also been suggested that nanobubbles could be clinically safe in human; because the filter pore size of cardiopulmonary bypass machines are usually in the range of 28–40 µm, nanobubbles of <10 µm in diameter are therefore negligibly small as a causal substance of gas embolism in blood vessels 2 in blood of fishes and mice are not monitored. As we have not used other gas-nonobubble water, whether promoting effect on growth is due to nanobubbles themselves or elevated oxygen concentration in the water is still unresolved. Further examination using other gas nanobubbles is required to determine the effect of nanobubbles themselves on growth of lives. Although there are several limitations, air and oxygen-nanobubble water significantly promoted the growth of plants, fishes, and mice, and this novel finding may bring new insight in effective growth of lives in the future.There are several limitations in this study. In fishes and mice experiment, food intake was not controlled. The promoting effect of air and oxygen-nanobubble water on growth may be partially due to increase in oral uptake. In addition, although previous study have demonstrated that oxygen-nanobubble saline effectively improved hypoxic conditions of swine blood ex vivo


Background. gcrR gene acts as a negative regulator related to sucrose-dependent adherence in S. mutans. It is constructive to test the potential capacity of mutans with gcrR gene deficient in bacteria replacement therapy. Methods. In this study, we constructed the mutant by homologous recombination. The morphological characteristics of biofilms were analyzed by confocal laser scanning microscopy. S. mutans UA159 and the mutant MS-gcrR-def were inoculated, respectively, or together for competitive testing in vitro and in rat model. Results. Adhesion assay showed that the adhesion ability of the mutant increased relative to the wild type, especially in the early stage. MS-gcrR-def out-competed S. mutans UA159 in vitro biofilm, and correspondingly coinfection displayed significantly fewer caries in vivo. The former possessed both a lower level of acid production and a stronger colonization potential than S. mutans UA159. Conclusion. These findings demonstrate that MS-gcrR-def appears to be a good candidate for replacement therapy. Dental caries is a well known major oral health problem in most countries. According to a national survey of the oral health status in China in 2005, more than 50% of children aged 7 years were affected by dental caries . AlthougStreptococcus sanguis [Streptococcus gordonii [Streptococcus salivarius [Streptococcus mutans  and failed in replacement therapy. The attempts to search an appropriate strain to prevent caries became focused on gene-modulation for the main pathogen, MS.The concept of bacteria replacement therapy against dental caries has been put up early in the middle of last century , related sanguis , Streptogordonii , 5, and livarius . UnfortuS. mutans is recognized as the main pathogen of dental caries in humans. In the procession development of caries, the paramount main virulence factor involves the ability to produce acid and adhere to the tooth surface [S. mutans to the tooth surface prevents the bacteria from being washed away by chewing or by the flow of saliva. The rationale of the sucrose-dependent adherence can be used by the low acid-producing strains to enhance their adherence ability. Thus, these strains occupy the same ecological niche in plaque similar to their more cariogenic progenitor [gcrR knock out, a gene regulated with sucrose-dependent adherence of S. mutans, called GMS900 [ surface . In partogenitor . Idone gS. mutans mutant and confirmed its ability of replacement therapy both in vitro and in vivo. This study demonstrated that the mutant strain, which lacks the whole open reading frame (ORF) of the gcrR gene, had lower acid production and stronger colonization potential compared with the wild-type strain.In the present study, we constructed gcrR-deficient Escherichia coli was grown overnight at 37°C in Luria broth  with gentle aeration or on Luria agar plates supplemented with 100 μg/mL of ampicillin and/or kanamycin when appropriate . S. mutans UA159 was grown routinely as an overnight standing culture in brain-heart infusion (BHI) broth  at 37°C in 5% CO2. The mutants were selected on Todd-Hewitt (TH)  agar plates after the strains were incubated overnight at 37°C in 5% CO2. Kanamycin (500 μg/mL) was added to the growth medium to maintain the selective pressure for the MS-gcrR-def mutant.gcrR upstream and downstream (720 and 705 bp from S. mutans UA159) at the end of KanR, and was then purified using a PCR purification kit . The amplicon was then ligated to the pMD18-T simple plasmid to generate pMD-GK plasmid according to the manufacturer's instructions .The details of construction of gcrR-deficient mutant are in accordance with GMS900 generated by Idone [S. mutans UA159 cells were transformed with the final pMD-GK plasmid in the presence of competence-stimulating peptide according to a previously described method [μg/mL of kanamycin after these colonies were incubated overnight at 37°C in 5% CO2.d method . TransfoS. mutans wild-type and mutant strains were monitored to determine their adherence to the walls of 24-well cell culture plates following 2, 4, and 6 h growths in BHI supplemented with 2% sucrose. In brief, overnight bacteria cultures were first standardized to 1.0 optical density (OD470) by using fresh physiological saline. Then the bacterial suspensions were diluted at a ratio of 1 : 20 with fresh BHI that contains 2% sucrose and distributed in triplicate in sterile 24-well cell culture plates  with 2.0 mL of fresh medium. The plate was kept at 37°C in 5% CO2 for 2, 4, and 6 h (in triplicate). Bacterial growth was determined at 470 nm by using a microplate reader. Crystal violet staining was used to monitor biofilm formation as described previously [eviously .S. mutans and MS-gcrR-def mutant standardized to 1.0 OD unit were diluted at a ratio of 1 : 20 in fresh BHI with 2% sucrose. The cells were then grown on glass coverslips placed in a sterile 6-well cell culture plate  in 5% CO2 at 37°C for 2, 4, 6, and 12 h. The biofilm samples were gently rinsed thrice with PBS to remove unattached cells, dried for 3 min, and stained with molecular probes SYTO 9  and propidium iodide . The stained samples were then examined via CLSM . Images were obtained from serial optical sections and captured at 488 nm. The Z section was used to record the biofilm thickness. The red and green fluorescence intensities were quantified using Image Pro-plus 6.0 .Wild-type S. mutans wild-type and mutant strains were diluted at a ratio of 1 : 20 in fresh BHI supplemented with 1% sucrose in triplicate. For in vitro acid production, the initial pH of the culture was measured at a constant pH of 7.3 by using a pH meter after the cell suspensions of S. mutans, MS-gcrR-def were mixed. The decrease in pH levels for 16 h was measured again by using a pH meter to obtain the ΔpH of these cultures.After the strains were standardized to 1.0 OD unit by using physiological saline, S. mutans UA159 cultures and the MS-gcrR-def mutant were inoculated in BHI with water (control treatment) or kanamycin and then standardized to 1.0 OD unit (OD470) by using fresh physiological saline. The two bacterial suspensions were diluted at a ratio of 1 : 20 with fresh BHI and distributed in 4.0 mL aliquots into sterile 6-well cell culture plates to obtain two-bacterial biofilm cells at 37°C for 4, 6, 8, 11, and 24 h. Each well was gently rinsed with distilled water to remove loose bacteria. Firmly attached cells were scraped from the substrate by pipetting and then rinsed with distilled water to ensure that all of the cells were collected. Total DNA was extracted from the same numbers of cells in the control group and the test groups by using a DNA extract kit . Total DNA was amplified in a TaKaRa SYBR Premix system. Amplification was performed using a 7500 real-time PCR system  according to the following thermal cycling protocol: initial denaturation at 95°C for 30 s and then followed by 40 cycles of the three steps . Standard curves were generated for each primer-template set with a series of standard S. mutans UA159 genomic DNAs  that were diluted 10-fold. The standard curves were then used to calculate the correlation coefficients and translate the concentration threshold (Ct) values into the relative DNA copy number. Ct is defined as the cycle in which SYBR green fluorescence is detected at a level above the background.Overnight 9 CFU/mL of S. mutans UA159, MS-gcrR-def or mixture of those two strains in ratio of 1 : 1 per mL. Over the following 3 days, the rats were challenged repeatedly with fresh swabs of the appropriate strain, and on the next day after challenge, swab samples were collected and plated onto MSB agar plated to confirm colonization. All the groups were provided caries-promoting diet [Eighty specific pathogen-free (SPF) female SD rats  at 18 days of age were chosen for the in vivo study. In order to facilitate the cariogenic bacterial planting, all the rats were fed with appropriate antibiotics  for three consecutive days. these groups of six rats each were challenged with oral swabs saturated with 2 × 10ing diet . Forty dt-test. P value < 0.05 was considered statistically significant.Experimental data were analyzed on SPSS 16.0 software  and presented as means ± standard deviation. Two-group comparisons were performed using Student's gcrR gene and the replacement with the KanR cassette as a result of double-crossover recombination. Chromosomal DNA was isolated from the selected transformants and UA159 progenitor, which was used as a template for PCR amplification with the P1/P2 and RT-gcrRF/RT-gcrRR primers (gcrR gene) were analyzed via agarose gel electrophoresis and their sizes were compared with those predicted after a successful allelic exchange event occurred . Accordingly, at the early stage (at 2 h and 4 h) MS-gcrR-def formed biofilms more quickly than UA159 and the biofilms were thicker relative to the wild type, which demonstrated MS-gcrR-def is a potential stronger adhesive to preempting tooth surface.Biofilm structures were analyzed by CLSM. The top view and vertical section of the biofilm by ectively . 2 h andldh in metabolism .To explore potential mechanisms of hypocariogenicity of MS-gcrR-def in competition with UA159, we examined the capacity of acid production of both strains, as well as the dominate gene tabolism . The iniTo certify the dominance in dual-species biofilm in replacement therapy, we inoculated the two strains into polystyrene plates with identical initial condition. The mutant exhibited an advantage over the wild type when these strains were cultured together. In particular, the gcrR-defective bacteria accounted for >90% in all of the adherent bacteria . Thus, tP < 0.05). There was no significant difference in dental caries lesions between dual-bacteria group and MS-gcrR-def monospecies group (P > 0.05).Since there is conspicuous superiority of MS-gcrR-def in the dual-bacteria biofilm, the mixing bacteria model in germfree rats is constructed to confirm its hypocariogenic ability in vivo. The rats are sacrificed 40 days after initial inoculation. The depth of carious lesion in both maxillary and mandibles involvement was scored for statistics . The dueIn the present study, we addressed the relationship both in vitro and in vivo of two strains, UA159 and MS-gcrR-def, and indicated a potential effector strain in bacterial replacement therapy. It was introduced to us with the increasing desire for natural products for health maintenance. For many years, replacement therapy has been considered a promising approach for the prevention of certain microbial infections . ParenteS. mutans is by far the most common pathogen found in dental caries and may get spontaneous mutation in environmental induction. Directly implant the harmless bacteria which are strongly competitive with S. mutans, leading to achieving tailor-made protection for the host against it. Since the effector strain is closely contacted to the host, herd protection through natural transmission might be increased too. Jeffrey Hillman's group generate a hypocariogenic strain which affects the acid production of S. mutans by using recombinant DNA methodology and persistently continue the research for 20 years [S. mutans to develop the effector strain with ecological volume exclusion and low acid production.20 years . Mutant 20 years . In thisα-1,6-glycosidic linkages, is ubiquitous on the S. mutans surface. GBL is an adhesin and can be isolated from many S. mutans subtypes [S. mutans can synthesize many kinds of glucan to form a wrap of mucosal fluid, which promotes its tight adherence to the tooth surface. Although different opinions on the mechanism by which GBL functions have been presented, GBL has a very important function in the formation of dental plaque and adherence of S. mutans [gcrR gene functions as a negative transcriptional regulator of the gbpC gene, which encodes the S. mutans GBL. Some researchers revealed that the gcrR expression product shares a high homology with the signaling molecule of the density inductance system, which may be involved in the regulation of the GBL expression. For instance, Sato et al. [gbpC gene by gcrR gene. On the contrary, the gcrR gene knockout results in the overexpression of GBL. In the study of Idone et al., RT-PCR demonstrated an increase in gtfD, gbpC gene of strain GM900, leading to augment in sucrose-dependent adherence. Our group did the same research on MS-gcrR-def and got the identical result (data not shown). Interestingly, they suggested the aggregate of GM900 increase in vitro, while it reveals compromise in their ability to colonize in vitro model of tooth surface and to rat molars than the wild type. We have the converse result to the point. In our research, comparing with UA159, MS-gcrR-def had evident superiority in sucrose-dependent adherence to borosilicate glass and absolutely dominated in the biofilm construction, indicating strong competitiveness of the former by the weight of evidence. It may be due to the strain of S. mutans and culture method.Glucan-binding lectin (GBL), which enables bacteria to aggregate by binding the subtypes . With th. mutans . The gcro et al.  revealedgcrR results in consecutive descent in pH value of MS-gcrR-def cells, as well as the biofilm it forms. In 2008, Dunning and colleagues showed that GMS901, the S. mutans mutant, which harbors insertion-deletion mutation in gcrR, is acid sensitive and has a significantly lower acid tolerance response (ATR) compared with the wild-type progenitor strain S. mutans UA159 [gcrR could increase the expression of gene ffh and gene atpA/E which related to ATR and may modulate the pH value through proton-translocating ATPase on the cell membrane [In this case, the deficiency of gene ns UA159 . They sumembrane .Taken together, MS-gcrR-def showed reduced acid production and significantly enhanced adhesion ability at the early stage, which might confer it an absolute advantage in the competition with wild type. These findings suggest that MS-gcrR-def is a promising effect strain in place of UA159. In the replacement therapy, even if the wild-type strain interference cannot be avoided, the mutant, with its strong early colonization ability, is still able to become the dominant bacteria on the surface of tooth and reduce the incidence of dental caries.


Background: The bacterial Isc operon contains a ferredoxin whose precise role is unknown and a desulfurase enzyme.Results: We have structurally characterized the complex of Escherichia coli ferredoxin with the desulfurase IscS.Conclusion: We show that ferredoxin occupies a groove close to the active site.Significance: Our results shed light into the mechanism of iron-sulfur cluster biogenesis. isc) operon is an essential machine that is highly conserved from bacteria to primates and responsible for iron-sulfur cluster biogenesis. Among its components are the genes for the desulfurase IscS that provides sulfur for cluster formation, and a specialized ferredoxin (Fdx) whose role is still unknown. Preliminary evidence suggests that IscS and Fdx interact but nothing is known about the binding site and the role of the interaction. Here, we have characterized the interaction using a combination of biophysical tools and mutagenesis. By modeling the Fdx·IscS complex based on experimental restraints we show that Fdx competes for the binding site of CyaY, the bacterial ortholog of frataxin and sits in a cavity close to the enzyme active site. By in vivo mutagenesis in bacteria we prove the importance of the surface of interaction for cluster formation. Our data provide the first structural insights into the role of Fdx in cluster assembly.The bacterial iron-sulfur cluster ( Iron-sulfur (Fe-S) clusters are essential prosthetic groups that provide an important source of redox potential to the cell. They are usually coordinated to proteins by cysteines and histidines, sometimes complemented by aspartic groups. Fe-S cluster proteins are ubiquitous and perform a variety of roles including electron transfer, enzyme regulation, and regulation of gene expression , 2. Becal-cysteine to l-alanine, and S0, forming a highly reactive persulfide on the catalytic cysteine. S0 is subsequently transferred as S2− to the scaffold protein, and along with Fe3+, is coordinated by the thiolate ligands of three highly conserved cysteine residues and forms a [2Fe-2S] cluster  –8. They  cluster . Generat cluster . Along w enzymes –13. In Fchondria –12.Escherichia coli Fdx was shown to interact with IscS, HscA, and IscA by co-purification , mutagenesis, and small angle x-ray scattering (SAXS). We also tested the interaction in the presence of IscU and the bacterial ortholog of frataxin, CyaY, whose complexes with IscS have been previously characterized (in vivo by mutagenesis studies in E. coli.To gain new insights into the role of holo-Fdx in Fe-S cluster assembly we have characterized the structure of the cterized –20. We fE. coli fdx was amplified by PCR from E. coli genomic DNA with a 5′ NcoI restriction site and a 3′ stop codon and NotI restriction site and cloned into a modified pET-24 vector (EMBL Hamburg) with a GST tag, His tag, tobacco etch virus protease cleavage site and NcoI restriction site in tandem upstream of the NotI restriction site. IscS mutants were prepared by site-directed mutagenesis of the construct pET-11 IscS (EMBL-Hamburg) (The DNA of Hamburg)  using Qu280 = 6,990 m−1 cm−1. The amount of holo- with respect to apo-Fdx in the sample was estimated by taking the A458/A280 value of >0.45 to be >90% holo-Fdx . His-tagged tobacco etch virus protease (in-house) was used to cleave the N-terminal GST/His tag under dialysis overnight at 4 °C. The reaction mixture was passed through a nickel-nitrilotriacetic acid-agarose gel and further purified using a 16/60 Superdex G75 column  followed by ion exchange using a MonoQ HR 5/5 column (Pharmacia Biotech). Protein concentration was determined using ϵholo-Fdx .E. coli CyaY, IscU, IscS, and its mutants were expressed and purified as described previously (15N- and 15N/13C-labeled proteins were expressed in E. coli BL21(DE3) in M9 minimal medium prepared with [15N](NH4)2SO4  and/or d-[13C]glucose . For [15N/2H]Fdx the growth medium was prepared with 99% deuterium oxide  and d-[12C/2H]glucose .Unlabeled eviously , 23–26. 15N]- or [15N/2H]Fdx or Fdx mutants were titrated with IscS, IscS mutants, IscU and/or CyaY, all in NMR binding buffer phosphine, pH 8) at 298 K. 15N-SOFAST HMQC spectra .  cluster of holo-Fdx is oriented between the active site cysteine loop of IscS and the cysteine ligands of IscU. The C terminus of holo-Fdx, which contains a histidine and a tyrosine, points toward the interface between IscS and IscU, and could be involved in electron transfer between the [2Fe-2S] cluster of holo-Fdx to the active site cysteine loop of IscS and/or the [2Fe-2S] cluster on IscU. This model provides the first structural insights into the holo-Fdx complex and its importance for cluster formation.Holo-Fdx binds in a cleft between the two IscS protomers A and covrostatic B, involvith CyaY  in agreeith CyaY C. In sucin vivo assay to test the importance of the residues involved in Fdx·IscS interaction in Fe-S biogenesis. We employed the E. coli Fe-S cluster-dependent transcriptional regulator IscR as reporter for Fe-S protein maturation and used an E. coli strain carrying the lacZ reporter gene fused to a gene whose expression is repressed by the Fe-S bound form of IscR, iscR (PiscR::lacZ)  . As prevpression  37). Th. Thin viK mutant . These rin vivo assay in E. coli.Identification of the machine that is responsible for iron-sulfur cluster biogenesis dates relatively recently . First dfdx strain suggests that Fdx Asp74 might be more important than Asp70 for the interaction with IscS and/or other binding partners. This is consistent with reports that demonstrate the importance of Asp74 in the primary interaction domain of human and bovine adrenodoxin ” ” . While tr both)” . Our resclusters , 10, 46.Some open questions remain that will need further investigations. We observe that Fdx competes for the same binding site that accommodates CyaY, whereas leaving the interaction with IscU unchanged. In addition, the Fdx binding site overlaps not only with CyaY but also with at least two other proteins, YfhJ (IscX), part of the ISC operon, and TusA . IscS fo


Osteosarcoma is the most common malignant bone tumor in children and adolescents. This aggressive cancer mostly occurs in the long bones. Therefore, novel therapeutic approaches, such as biological therapies and gene therapy, are required to efficiently treat osteosarcoma. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) has been demonstrated to inhibit the growth of several types of cancer cells and a number of studies have shown that osteosarcoma may be vulnerable to biological therapies. However, little is known regarding the therapeutic effects of capsaicin on osteosarcoma. This study investigated the effects of capsaicin on MG63 human osteosarcoma cells, in addition to elucidating the regulatory signaling pathways underlying the effects of capsaicin, the caspase cascade and the antioxidant enzyme system. The MG63 cell line was treated with various concentrations of capsaicin. Cells were analyzed using MTT and flow cytometry, and the presence of DNA fragmentation was evaluated using TUNEL assay. Results showed capsaicin induced apoptosis in MG63 cells. Thus, capsaicin exhibited an anticancer effect in osteosarcoma cells. Capsicum and a chemiluminescence (ECL) kit was obtained from Amersham Pharmacia Biotech . Bcl-2, Bcl-2-associated X protein (Bax) and pro-caspase-3 were obtained from Epitomics, Inc. , while phosphorylated extracellular signal-regulated kinase (p-ERK), phosphorylated p-38 (p-p38) and phosphorylated c-Jun N-terminal kinase (p-JNK) were purchased from Cell Signaling Technology, Inc. .Capsaicin and MTT were purchased from Sigma-Aldrich , while Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies . The U0126, PD98053, SP600125 and Z-VAD-FMK used in the study were purchased from Calbiochem2 incubator and cultured for 2–4 days until confluence was reached. Subcultures were prepared using 0.05% trypsin solution and seeded in 6- or 96-well tissue culture plates. Serum was starved from the culture media at the time of adding various agents.MG63 cells (human osteosarcoma cell line) were purchased from the Korean Cell Line Bank  and cultured in DMEM containing 10% heat-inactivated FBS. The cells were plated in tissue culture dishes at 37°C in a humidified 5% CO2 cells per well and indicated concentrations of capsaicin were added for indicated time-periods. Briefly, following treatment with capsaicin at various concentrations  and various time-periods  under 150 μM of capsaicin, the cells were washed and 0.5 mg/ml MTT in DMEM solution was added to each well, prior to incubation for 2 h at 37°C. The supernatant was then removed and the cells were dissolved in dimethylsulfoxide (DMSO). The absorbance of each well was measured at 570 nm with a 680 microplate enzyme-linked immunosorbent assay (ELISA) reader .Cell viability was assessed using an MTT assay, based on the reduction of MTT into formazan dye by the action of mitochondrial enzymes. MG63 cells were seeded in 96-well plates at a density of 5×104 cells per well and incubated for 24 h with 50–400 μM capsaicin . Cell morphology was examined under a light microscope.The untreated and treated cells were seeded in 6-well plates at a density of 5×104 cells per well and treated with the indicated reagents for 24 h at 37°C. The suspended and adherent cells were then harvested using 0.05% trypsin solution. The harvested cells were centrifuged at 10,000 × g for 15 min at 4°C and the pellets were then washed in PBS, prior to the addition of fixing solution with ice-cold 100% ethanol containing 0.25% Triton X-100 for treatment overnight at 4°C. Subsequent to fixation, the cells were washed and stained with 50 μg/ml propidium iodide containing 100 μg/ml RNase, prior to being incubated for 20 min at 37°C and analyzed using a FACSort flow cytometer .Cells were seeded in 6-well plates at a density of 5×10in situ Cell Death Detection kit (fluorescein) from Roche Applied Science . The cells were seeded in cover slides (5×102 cells per slide) and then treated with capsaicin. Following this, the cells were washed in PBS and freshly prepared 4% paraformaldehyde was added for cell fixation for 1 h at 37°C in a humidified 5% CO2 incubator. The cells were then washed again in PBS, prior to being permeabilized in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. The cells were then subjected to the TUNEL reaction at 37°C in a humidified atmosphere in the dark for 60 min. The fluorescence signal emitted by the fluorescein-labeled dUTP incorporated into the fragmented DNA was detected using Leica confocal microscopy .The presence of DNA fragmentation was evaluated using TUNEL assay with an Capsaicin-treated cells were harvested using 0.05% trypsin solution and then suspended with 0.4% trypan blue solution. The cells were counted using a hemocytometer under a light microscope and cells that were observed to exclude the dye were considered viable.4 cells/cm2, cultured and incubated in DMEM containing 10% FBS. Prior to treatment with the indicated conditions, the cells were serum-starved overnight, treated with the agent and then harvested. Using lysis buffer , the cells were lysed on ice. The lysates were subsequently centrifuged at 10,000 × g for 20 min at 4°C and the supernatants were loaded on 15% sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel and transferred to a nitrocellulose membrane. The membranes were subsequently immunoblotted with various primary antibodies and incubated with the respective peroxidase-conjugated secondary antibodies. The signals were visualized using an enhanced ECL kit from Amersham Pharmacia Biotech.The cells were seeded in 6-well plates at a density of 5×10Experiments were performed at least three times. Statistical significance was analyzed using a Student's t-test (two-tailed). P<0.05 was considered to indicate a statistically significant difference.We examined the effects of capsaicin in the MG63 osteosarcoma cell lines, which had been treated with various concentrations of capsaicin  for various time-periods . As shown in MG63 cells were cultured for 24 h with different concentrations of capsaicin . Following 24 h treatment with capsaicin, no significant morphological changes were observed in the cells treated with capsaicin at 50 and 100 μM. However, the cells exhibited the morphological features of apoptosis when treated with 150 μM capsaicin for 24 h . These mIn order to characterize the type of cell death that had been observed, we examined whether the cell death was apoptosis. MG63 cells were treated with 150 μM capsaicin for 24 h and the apoptotic DNA fragmentation of the MG63 cells was visualized using TUNEL assay. TUNEL assay is a tool that is frequently used for the detection of DNA fragmentation ,19. The To investigate the effect of capsaicin on protein molecules that are involved in apoptosis, western blot analysis was used to test for the presence of the anti-apoptotic proteins Bcl-2 and cleaved caspase 3 (pro-caspase-3) and the pro-apoptotic protein Bax. MG63 cells were treated with various concentrations of capsaicin for 24 h and with 150 μM capsaicin for different time-periods. Capsaicin decreased the expression of pro-caspase-3 and Bcl-2, while the expression of Bax was increased in a dose- and time-dependent manner .It has been demonstrated that apoptosis leads to various signaling processes and, among them, the mitogen-activated protein kinases (MAPKs) , the casIn this study, we used a variety of techniques and demonstrated that the cell viability of MG63 cells was able to be reduced by capsaicin in a dose- and time-dependent manner. In addition, the capsaicin-treated group showed an increased level of positivity with the TUNEL assay and an increase in Bax protein. Moreover, the experiments with the signaling pathway inhibitors showed that the groups pretreated with Z-VAD-FMK, NAC and catalase, respectively, had different results compared with those from the group treated only with capsaicin. These results indicated that the capsaicin-induced apoptosis in MG63 cells may have been mediated by the caspase cascade and the antioxidant enzyme system, among various signaling pathways.Osteosarcoma is the most frequently occurring primary malignant neoplasms of the long bones, including the distal femur and the proximal humerus, and mainly affects children and adolescents ,23. The Capsicum, has long been used in drugs for weight loss and has been studied as an attractive drug for cancer treatment, as an agent that induces apoptosis in various cell types in vitro(in vivo as the mechanism of tumor cell elimination in animal models for carcinogenesis (It has been demonstrated that a number of reagents are able to induce apoptosis on MG63 human osteosarcoma cells –27; howe in vitro–31. Moreogenesis ,33. ThesApoptosis has been suggested as a promising target for cancer chemotherapy –36. ApopThis study was designed to investigate capsaicin-induced apoptosis in MG63 human osteosarcoma cells and its underlying molecular mechanisms. Using TUNEL assay, flow cytometric assay and western blot analysis, we demonstrated that the anticancer effect of capsaicin resulted in morphological changes, decreased cell viability and apoptosis in the MG63 cells –4. TheseWe investigated the molecular factors that were involved in the apoptosis of capsaicin-treated MG63 cells. The MAPKs are expressed in all mammalian cell types and have individually different functions in the regulation of specific cell responses. MAPKs have been demonstrated to be composed of three parallel kinase modules, including ERK, JNK and p-38-MAPK –44. As sCaenorhabditis elegans, the cell death gene, CED-3 (It has been revealed that caspase, or cysteine-aspartic protease, belongs to the group of enzymes known as cysteine proteases, which are homologous to the e, CED-3 ,49. Cyste, CED-3 ,51. Moree, CED-3 . Furthere, CED-3 . Our rese, CED-3 .In present study it was demonstrated that the antioxidant enzyme system was also involved in the capsaicin-induced apoptosis. The antioxidant enzyme system has been indicated to be important in the control of apoptosis ,55. In ain vitro and in vivo studies are required before capasaicin is able to be ultimately applied to treat human patients with osteosarcoma.The present study provided distinct results describing the effect of capsaicin on MG63 cells, in addition to elucidating the molecular mechanisms that were implicated in the indution of apoptosis. In combination, the results showed that capsaicin induced apoptosis in the MG63 cells and that the caspase cascade and antioxidant enzyme system were the underlying regulatory signaling pathways involved in the capsaicin-induced apoptosis. In a previous study, we demonstrated the effect of capsaicin on human glioblastoma U87MG cells and concluded that capsaicin induced apoptosis in the U87MG cells . The pre


Here, the expression levels of these candidate miRNAs were determined in peripheral blood mononuclear cells (PBMCs) and the serum of MS patients, in addition to three genotyped single nucleotide polymorphisms (SNPs). Mapping in the genomic regions of miR-223, miR-23a and miR-15b genes, 399 cases and 420 controls were tested. Expression levels of miR-223 and miR-23a were altered in PBMCs from MS patients versus controls. Conversely, there were no differences in the expression levels of miR-15b. A significantly decreased genotypic frequency of miR-223 rs1044165 T/T genotype was observed in MS patients. Moreover, the allelic frequency of miR-23a rs3745453 C allele was significantly increased in patients versus controls. In contrast, there were no differences in the distribution of miR-15b SNP. In conclusion, our results suggest that miR-223 and miR-23a could play a role in the pathogenesis of MS. Moreover, miR-223 rs1044165 polymorphism likely acts as a protective factor, while miR-23a rs3745453 variant seems to act as a risk factor for MS.Evidence underlines the importance of microRNAs (miRNAs) in the pathogenesis of multiple sclerosis (MS). Based on the fact that miRNAs are present in human biological fluids, we previously showed that miR-223, miR-23a and miR-15b levels were downregulated in the sera of MS patients  Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS) among young adults. The cause of MS is not clear, as the disease develops in genetically susceptible individuals with the contributions of environmental factors, such as infection, sunlight exposure, and vitamin D deficiency . The climiRNAs are small noncoding RNA molecules that post-transcriptionally regulate gene expression by targeting the 3′ untraslated region (3′ UTR) of specific messenger RNAs (mRNAs). miRNAs play important roles in various biologic processes such as cell proliferation, development, differentiation, metabolism, apoptosis, angiogenesis, inflammation and immunity –11.Recent evidence showed the presence of stable miRNAs in biological fluids, such as blood, serum, plasma and cerebrospinal fluid (CSF) ,13. The versus healthy controls [In our previous study, we showed that expression levels of miR-223, miR-23a and miR-15b were downregulated in the sera of MS patients The ease with which blood can be obtained in a manner that is minimally invasive to the patient encouraged us to go further in the analyses of miR-223, miR-23a and miR-15b in the cells of this tissue.In particular, we determined the expression levels of these miRNAs both in PBMCs and sera from MS patients in order to establish a possible correlation between the levels of miR-223, miR-23a and miR-15b inside and outside the blood cells. Moreover, based on the fact that genetic alterations could influence miRNA expression and possibly play a role in disease susceptibility, we genotyped three SNPs, mapping in the genomic regions of miR-223, miR-23a and miR-15b genes.versus controls led miRNAs to be considered the new potential prognostic biomarkers for MS [In the past few years, the identification of miRNAs differently expressed in blood and lesions of MS patients s for MS . This ids for MS ,13. Secrs for MS .versus controls. In particular, miR-223, miR-23a and miR-15b levels were significantly reduced [We previously performed an analysis of circulating miRNAs in sera of MS and healthy control subjects, finding a general downregulation of the expression levels of serum miRNAs in MS patients vs. 0.49 ± 0.12, p < 0.02, versus controls  but not in PPMS patients .Correlation analysis between miRNA levels in PBMCs or serum and age or sex were carried out, and no statistically significant correlations were found  block in which each gene is located. Although the SNPs we identified have not been found in previous Genome Wide Association Study (GWAS), it is not known whether they were included in any of the arrays used. However, our choice was driven by a candidate SNP hypothesis, i.e., genetic alterations in candidate miRNA-encoding genes may, to some extent, influence miRNA function, as previously shown in other contexts . Conversely, a significant increased allelic frequency of miR-23a rs3745453 C allele in patients with MS as compared with controls was observed . No differences in allelic and genotypic distribution of miR-15b rs1451761 SNP were observed between patients and controls (p > 0.05). No significant correlations were found between these SNPs and miRNA expression levels (p > 0.05). The analysis of the three tagging SNPs revealed that the miR-223 rs1044165T allele likely acts as protective factor, whereas the miR-23a rs3745453C allele seems to exert a risk factor for MS pathogenesis. Interestingly, we found that the miR-223 gene is in LD with the VISG4 gene, a B7 family-related protein V-set and Ig domain-containing 4. VISG4 is a negative regulator of T cell activation. Vogt et al. demonstrated that VISG4 is a strong inhibitor of T cell proliferation in vitro and in vivo. The restricted expression of this protein on resting tissue macrophages suggests that VISG4 has an important role in the maintenance of T cell unresponsiveness in healthy tissue [VISG4 gene in LD with miR-223 locus would be responsible for the association found with rs1044165 in our MS population. However, a deeper investigation of genetic variability in the VISG4 gene is required to confirm our hypothesis.Allele and genotype frequencies of y tissue . Thus, aThis study was approved by the Institutional Review Board (IRB) of the University of Milan, Fondazione Cà Granda, IRCCS Ospedale Maggiore Policlinico. All subjects provided written informed consent.For the genetic analysis, the MS population was recruited at the Multiple Sclerosis Center of Fondazione Cà Granda IRCCS Ospedale Maggiore Policlinico and at the AOU S. Giovanni Battista di Torino. The female-to-male ratio was approximately 2:1 ; the mean age was 43.9 ± 0.7 years; the mean age of onset was 32.4 ± 0.6 and the mean disease duration was 12.9 ± 0.6. All patients underwent the standard work up for MS ) and diagnoses were based on the McDonald’s criteria . Four-huThe characteristics of MS patients and controls enrolled for the expression analysis were summarized in High-molecular weight DNA was isolated from whole blood using a Flexigene Kit , as described by the manufacturer. The amount of DNA for each sample was determined by measuring the optical density at 260 nm wavelength using a spectrophotometer . DNA samples were aliquoted and stored at −20 °C until used.g at room temperature for 15–20 min. The serum was removed and dispensed in aliquots of 400 μL into cryo-tubes. Specimens were stored at −80 °C.Whole blood samples were allowed to sit at room temperature for a minimum of 30 min and a max of 2 h, after collection. Separation of the clot was done by centrifugation at 1000–1300× Total RNA enriched for small RNA species was isolated from PBMCs, using the mirVANA miRNA Isolation Kit according to the instruction of the manufacturer , and the RNA concentration determined by using a NanoDrop ND-3300® Fluorospectrometer.C. elegans miRNAs cel-miR-39 (Qiagen) were added  to each denatured sample [Four-hundred microliters of human serum was thawed on ice and lysed with equal volume of 2× Denaturing Solution . To allow for sample-to-sample normalization, synthetic d sample . RNA was2), which was the normalized gene expression ((−Delta Ct)2) in the MS group divided by the normalized gene expression ((−Delta Ct)2) in the control group, as previously described [p value cut off by t-test is p < 0.05.The reverse transcription reaction was performed using the TaqMan@MicroRNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions. The quantitative Real-time PCR was performed on the ABI 7500 FAST system (Applied Biosystems). miR-223, miR-23a and miR-15b were detected with single TaqMan® miRNA assay (Applied Biosystems). Data were analyzed with SDS Relative Quantification Software version 2.2.2 , with the automatic Ct setting for assigning baseline and threshold for Ct determination. Relative expression levels were calculated as Fold-Change ((−Delta Delta Ct)escribed ,27. The http://hapmap.ncbi.nlm.nih.gov/) and Ensemble browser (http://www.ensembl.org/index.html) to determine the position of miR-223, miR-23a and miR-15b genes and Haploview software (http://www.broad.mit.edu/mpg/haploview/) to identify the tagging SNPs of miR-223, miR-23a and miR-15b genes . The SNPs were analyzed using the TaqMan methodology, according to the instructions of the manufacturer.We used Hapmap project (http://www.husdyr.kvl.dk/htm/kc/ popgen/genetik/applets/kitest.htm). Both cases and controls were in HWE. Chi Square test was used to test for differences in allele distributions between the groups. Odds ratio (OR) was calculated along with its 95% Confidence Interval (CI).Statistical analysis was performed using the Sigma Stat 3.1 software . Allelic and genotypic frequencies were obtained by direct counting. Chi square test was used to test for Hardy Weinberg Equilibrium (HWE, Expression levels of miR-223, miR-23a and miR-15b were analyzed using the One-Way Anova test.In this study, we found different expression levels of miR-223 and miR-23a in PBMCs of MS patients compared to controls, suggesting a possible involvement of these two miRNAs in the pathogenic mechanisms of the disease. In particular, we observed a significant upregulation of miR-223 and miR-23a levels in RRMS, but not in PPMS patients, though this could be due to the small number of PPMS cases. Therefore, we are planning to increase the number of PPMS patients in order to confirm our hypothesis.miR-223 SNP as a protective factor, and miR-23a variant as a risk factor for MS. However, genetic analysis on a larger population is required to draw definitive conclusions.The genetic analysis showed


Motor deficits were observed in all models, with histological analysis of tyrosine hydroxylase positive cells in the SNpc revealing a significant loss of DAergic neurons in all animal models. In addition, the α-syn animal model also presented a reduction in exploratory activity, and the 6-OHDA and PQ animals displayed a significant increase in both depressive- and anxiety-like behavior. Interestingly, cognitive impairment (working memory) was only observed in the 6-OHDA model. Overall, these PD models are suitable for mimicking the motor symptoms associated to PD, with each encompassing other relevant NMS components of the disorder that may prove beneficial for further studies in PD.Parkinson's disease (PD) is classically characterized by motor symptoms; however, non-motor symptoms (NMS) are increasingly recognized as relevant in disease-state, given the associated alterations in mood (depression and anxiety) and cognition. Here, particularly in regards to NMS, we aimed to compare the motor, emotional and cognitive behavior of three animal models of PD that trigger dopaminergic (DAergic) degeneration on both brain hemispheres: (i) the 6-hydroxydopamine  lesion model; (ii) the paraquat (PQ) induced model, and (iii) a genetic model based on α-synuclein overexpression (α-syn). 6-OHDA and α-syn vector were injected bilaterally in the  This degeneration of DAergic nigrostriatal system is largely responsible for the classical motor signs, including: resting tremor, muscle rigidity, and bradykinesia , one of the most frequent neurodegenerative disorders, is characterized by the progressive loss of dopamine (DA) neurons in the Of particular concern, NMS can include emotional and cognitive deficits, sleep disorders, and autonomic, gastrointestinal, and sensory dysfunction  rat model has been the most extensively studied animal model of PD, including results from our group indicating that the unilateral 6-OHDA-lesion in the medial forebrain bundle is a suitable model to investigate depressive-like behavior and exploratory activity impairments associated with PD  or mutated α-synuclein (α-syn) in transgenic mice. In spite of providing a suitable model to study the involvement of α-syn in PD pathogenesis, this model present several limitations. In fact, none of the transgenic α-syn mouse models presented the key features of the disease, namely the extensive nigrostriatal DAergic degeneration or the motor impairments -mediated overexpression of α-syn) to assess emotional and cognitive behaviors, in parallel with motor performance. This phenotypic characterization was complemented with the immunohistological evaluation of the nigrostriatal lesion.Experiments were conducted in accordance with the national ethical requirements for animal research, and with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes. Young adult male Wistar rats  weighing 300–350 g at the beginning of the experiments, were housed in pairs, in appropriate cages, under standard controlled conditions . After surgeries, the animals were allowed to recover for 5 weeks before starting the behavioral assessment. Starting in the sixth week after surgery, all animals were exposed to the following sequential behavioral tests: elevated-plus maze followed by the forced swimming test (both performed in the sixth week), Morris water maze (seventh and eighth weeks), rotarod followed by open field and the sucrose preference test , and the skilled paw reaching test (week 11). The minimum interval between two consecutive procedures was 2 days. All animals were subjected to the same sequence of behavioral test and sacrificed 12 weeks after surgery. Rats were deeply anesthetized with pentobarbital and were transcardially perfused with 4% paraformaldehyde (PFA) in PBS. The brain was then removed surgically for immunohistochemistry (IHC) analysis.Under ketamine and medetomidine  anesthesia, the animals were placed on a stereotaxic frame. Animals were pre-treated with desipramine (25 mg/kg) 30 min prior to 6-OHDA injection in order to protect noradrenergic neurons. Bilateral injections of 8 or 6 μg of 6-OHDA  were performed in the SNpc  by meanUnder anesthesia with ketamine and medetomidine animals were implanted with Alzet mini-osmotic pumps  delivering for 28 days at a rate of 0.7 mg/day PQ (Sigma). Implants were placed in the subscapular region.11 genomes/μl (rAAV2-vector) and 5.5 × 1011 genomes/μl (rAAV2-α-syn). Each animal received 11 × 1011 genomes of the respective rAAV-vector.U6 promoter-driven α-syn expression system was established in AAV2 vector, with EGFP expression separately controlled by a CMV promoter as a marker for the transduction efficiency. The plasmid DNA vector only or AAV2-α-syn were co-transfected with plasmids pHelper and pAAV-RC to HEK293 AAV cells using a standard calcium phosphate method. At 72 h post-transfection, the cells were harvested and crude rAAV vector solutions were obtained by repeated freeze/thaw cycles. The cleared crude lysate was applied on a heparin column. After the total lysate passed through the column, the matrix was washed twice with 25 ml of PBS with low NaCl  and the virus eluted with 15 ml of PBS with high NaCl . The elute was concentrated to about 1 ml using a Millipore Biomax-100K NMWL filter device (UFV2BHK40) by centrifugation at 4000 rpm, 15–40 min. To adjust the NaCl concentration to physiological levels, the filter device was refilled with PBS (pH 7.4) and the virus concentrated to 250–300 μl. After removal of the virus-containing solution, the membrane of the filter device was washed three times with PBS (pH 7.4) that was added to the main part of the recombinant AAV2. The fractions containing high-titer rAAV vectors were collected and used for injection into animals. The number of rAAV genome copies was semi-quantified by PCR within the CMV promoter region using primers 5′-GACGTCAATAATGACGTATG-3′ and 5′-GGTAATAGCGATGACTAATACG-3′. The final titers were 6.4 × 10Anxiety-like behavior was measured in the elevated plus maze as previously described  as previously described ; shape and dimensions were similar to the device described by Montoya et al. . The app2O2. Sections were incubated with mouse anti-tyrosine hydroxylase (TH)  diluted in PBS with 1% FCS. Incubations with the primary antibodies were performed overnight at 4°C. Sections were then incubated for 1 h at room temperature with biotinylated secondary antibody, goat anti-mouse IgG , diluted in PBS. After that sections were incubated with avidin peroxidase  for 50 min at room temperature. The reaction product was visualized using diaminobenzidine  in TBS with 0.0024% H2O2.IHC was carried out in free-floating mesencephalic axial frozen sections (35 μm). The chromogenic IHC assay was used to detect DAergic neurons. Nonspecific antibody binding was blocked by incubation for 60 min in PBS with 10% FCS containing 0.1% Triton X-100. Endogenous peroxidase activity was quenched by 10 min incubation in 1% HN = VSNpc)( (ΣQ/ΣV(sect)); where N is the estimation of cell number, V is the total volume of SNpc, ΣQ is the total number of cells counted in the sections, and ΣV(sect) is the total volume of counted sections.The immunohistochemically processed sections were used to evaluate the total number of TH immunoreactive cells within the SNpc. On mesencephalic sections, the region comprising the TH positive cells, correspondent to SNpc, was delineated and the total number of TH positive cells was counted in the full extent of the structure per section. Every sixth section covering the entire extent of the SNpc was included in the counting procedure. The following formula was used to estimate the number of TH neurons: t-test, Mann–Whitney nonparametric test, one-way or repeated measures analysis of variance (ANOVA), as appropriate. Differences between groups were further analyzed by Dunnett's or Bonferroni's post tests. p-value of 0.05 was considered statistically significant. All statistical procedures were performed using GraphPad Prism, version 4 .All data sets were tested for normality distribution with the Shapiro–Wilk test prior to any statistical analysis. Data are expressed as mean ± standard error of mean (s.e.m.). Statistical analysis was performed using Student F(2.11) = 15.32; p = 0.0007] was observed in 6-OHDA model, when compared with sham rats, with post-hoc analysis showing a marked decrease in the number of TH positive cells in the SNpc of animals treated with both 8 and 6 μg of 6-OHDA . In the PQ-exposed rats a 39% reduction of nigral DAergic neurons was also observed . The less severe lesion (21%) was induced by the overexpression of α-syn .Histological analysis of TH positive cells in the SNpc revealed a significant loss of DAergic neurons in all the animal models Figure . The higt = 3.498 for PQ; U = 172.0 for α-syn; F(2.41) = 6.726 for 6-OHDA; all p < 0.01]  = 29.17; p < 0.001], but no effect for the factor lesion , as well as no interaction between these factors   = 21.66; p < 0.0001] and lesion  was verified, while no interaction was detected between these two factors   = 23.42; p < 0.001] and lesion , without interaction between the factors  .The skilled paw reaching test was use to assess forelimb use; this test was performed 10 weeks post-lesion and the number of pellets eaten was evaluated. Regarding the PQ model, ANOVA showed an effect for the factor time  Figure . In the ] Figure . Similar] Figure . Post-hoU = 548.398; p = 0.6334), number of ambulatory episodes , and number of rearings   accompanied by an increase in the rest time. Significant differences were also observed regarding the number of ambulatory episodes, with α-syn rats presenting lower number when compared with sham animals , and the number of rearings was also decreased   = 3.34; p = 0.0442]. A post-hoc comparison revealed that animals treated with 6-OHDA 8 μg traveled significantly less than sham rats. In contrast, 6-OHDA 6 μg rats traveled a total distance that did not differ from that observed in the sham group. No differences were observed in ambulatory episodes  and in number of rearings , in both 6-OHDA doses  = 5.203, p = 0.0101 for 6-OHDA]   = 1.799, p = 0.1765 for 6-OHDA]  Figure . No stat Figures .U = 70.0, p = 0.0028 and F(2.34) = 6.658, p = 0.0036, respectively] . Total immobility time was, however, only affected in the PQ model  and not in the 6-OHDA model    = 1.786, p = 0.1795 for 6-OHDA]   = 2.25; p = 0.0417] and a significant effect of time  , which was not observed in 6-OHDA rats. In high dose of 6-OHDA the improvements were observed between the first and second, and first and fourth days  whereas there was no difference between first and third day. Rats injected with low 6-OHDA dose only improved the performance between first and third day (p < 0.05). In the other tasks the escape latency and distance swam did not differ between the groups  (Figures The Morris water maze was performed to evaluate cognitive functions. The protocol was divided in three different tasks to investigate both working and spatial reference memories and cognitive flexibility. No interaction between lesion and time were found in PQ and α-syn models in any of the tasks (all  Figures . However] Figure . Post-ho Figures .The present work confirmed that all three animal models of PD trigger the characteristic motor symptoms of the disease, as a consequence of reduced number of TH-positive neurons in the SNpc. Furthermore, on the latter parameter, we found a range of variation from 20 to almost 50% in the reduction of TH-positive cells, which is within the ranges of variance observed in other studies using similar animals models (Kirik et al., The main goal of this study was, however, to characterize the NMS in each of these animal models. First, it is relevant to highlight, here animal models that triggered DAergic degeneration on both hemispheres were chosen, which is of importance to the emergence of the full spectrum of behavioral symptoms of the disease. Briefly, from analysis of an extensive battery of behavioral tests, the PQ, the 6-OHDA and the α-syn models of PD were shown to have distinctive patterns of NMS impairments.The use of the herbicide PQ to study PD and NMS is still emerging. In contrast, the study of motor deficits in this model has already been done both in rats (Somayajulu-Nitu et al., Injection of viral vectors in the SNpc, to promote the overexpression of wt or mutated human α-syn, has recently proved to be a promising model of PD. Confirming previous studies (Decressac et al., The most widely used model of PD consists in the injection of 6-OHDA into one of the following three target sites: SNpc, medial forebrain bundle or the caudate-putamen. In spite of being the most scrutinized, including NMS, there is still a lack of studies using the bilateral 6-OHDA lesion model. Here, rats injected with two doses of 6-OHDA, bilaterally into the SNpc, displayed motor deficits accompanied by several NMS; the latter including increased anxious and depressive-like behaviors, and working memory deficits. Surprisingly, no differences were observed between the two doses of 6-OHDA, suggesting that lower dose is sufficient to produce all the effects observed, being in accordance with the similar impact that both doses had on DAergic neurons degeneration.More specifically, the motor performance, assessed through the rotarod and skilled paw-reaching test, was clearly impaired. Motor abnormalities were also observed in the open field test, where 6-OHDA animals treated with higher dose displayed a shorter travel distance. In the elevated plus maze test, 6-OHDA treated animals showed a significant decrease in time spent in the open arms, while no differences in closed arms were observed, thus suggesting an anxious-like state in these animals. The anxious phenotype is in line with observations showing that infusion of 6-OHDA in the striatum also results in an increase in anxiety-like behavior (Tadaiesky et al., In conclusion, here the data demonstrates that all the models included in this study present the crucial requirements for a model of PD, namely a selective loss of DAergic neurons, together with detectable motor deficits. Nonetheless, in addition to the classical features of PD, these models displayed different patterns of NMS symptoms. Overall, the α-syn model proved to be useful to study changes in exploratory activity, while the PQ model offered a valuable approach to investigate neuropsychiatric dysfunction, specifically alterations in depression and anxiety-like behavior. Finally, the 6-OHDA model displayed the broader spectrum of NMS and thus might be of use to study in combination emotional and cognitive deficits associated to PD. In future studies, the intrinsic differences of these toxin and genetic models can be relevant to unravel the mechanism underlying NMS, as well as to screen drugs for symptomatic treatment of the disease.F. L. Campos performed surgeries, behavioral assessment, histological analysis, treatment protocols, data analysis and interpretation, and drafted the manuscript. M. M. Carvalho helped in surgical procedures and behavioral analysis. A. C. Cristovão helped in AAV-vector production and in revision of manuscript. G. Je produced the AAV-vector. G. Baltazar, A. J. Salgado, and Y. S. Kim helped in revision of the manuscript. N. Sousa contributed for data interpretation and for drafting, revision, and approval of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


It was previously reported that an association analysis based on haplotype clusters increased power over single-locus tests, and that another association test based on diplotype trend regression analysis outperformed other, more common association approaches. We suggest a novel algorithm to combine haplotype cluster- and diplotype-based analyses.Diplotyper combines a novel algorithm designed to cluster haplotypes of interest from a given set of haplotypes with two existing tools: Haploview, for analyses of linkage disequilibrium blocks and haplotypes, and PLINK, to generate all possible diplotypes from given genotypes of samples and calculate linear or logistic regression. In addition, procedures for generating all possible diplotypes from the haplotype clusters and transforming these diplotypes into PLINK formats were implemented.LIPC) gene polymorphisms or diplotypes and levels of high-density lipoprotein (HDL) cholesterol.Diplotyper is a fully automated tool for performing association analysis based on diplotypes in a population. Diplotyper was tested through association analysis of hepatic lipase (Diplotyper is useful for identifying more precise and distinct signals over single-locus tests. Causal mutations for health conditions with genetic bases can be identified through finding associations with haplotypes, a form of correlation known as linkage disequilibrium (LD) . InvestiP = 4.2 × 10-6 and P = 1.6 × 10-4, respectively)  in the promoter region are in almost perfect LD (R2 = 0.97) [2 = 0.92) in HapMap CEU (Caucasians of European descent from Utah) database [P = 8 × 10-10) [LIPC SNPs or diplotypes and HDL-C levels in a Korean population consisting of 7,536 individuals. = 0.97)  and bothdatabase . The intdatabase  and rs11database , and thedatabase  and rs10database , were asdatabase . The pro× 10-10) . We inveA haplotype cluster is defined as a set of haplotypes. A diplotype is defined as a haplotype cluster pair, the definition of which is extended from a haplotype pair. The diplotype is a homozygous diplotype or a heterozygous diplotype. LD blocks based on Gabriel et al.  or the FThe first step produces LD blocks and any haplotypes for these LD blocks adjusting a threshold frequency, using Haploview tool.The second step produces all possible haplotype pairs from SNP genotypes of samples employing PLINK, which implements the EM algorithm.H = {1, ..., HnH}, which is obtained in the first step. Another set, HS = {{1H}, ..., {nH}}, consists of subsets with a single element of H. Repeat frequency is initialized to 0. The procedure in this step is as follows.The third step starts with a set of haplotypes, Procedure: If the length of the HS subset is greater than the repeat frequency and none of the elements of the HS subset are equal to any element of H, and the last element of HS subset is less than each element of H, each element of H is added to HS subset. In this way, HS is updated and the repeat frequency increases by one. The procedure is repeated until the repeat frequency plus one is equal to the total number of H elements. Table H = {A, B, C, D}.A}/*" indicates a set of all haplotypes except the "A" haplotype. Therefore, the set includes not only the haplotypes with frequencies greater than a threshold, but also those with frequencies below a threshold.The fourth step produces the patterns of all possible diplotypes from the results of the third step. Table A}/*", consist of three possible diplotypes, "{A}{A}", "{A}*", or "**", just as the SNP alleles (A/B) comprise three genotypes . That is, input formats for PLINK can be made from the diplotypes of samples in a population. At this point, linear or logistic regression analysis can be carried out by PLINK.In the final step, the diplotypes from samples are transformed into AA, AB, or BB formats according to the patterns of the diplotypes generated in the fourth step. For example, the diplotypes, "{http://code.google.com/p/diplotyper/downloads/list.Diplotyper is freely available on the website The population data used in the present study was provided by the Korea Association Resource (KARE) project from the Korean Genome Epidemiology Study (KoGES), which is conducted by the Korean National Institute of Health (KNIH). A cross-sectional analysis of samples from urban and rural communities in Korea was conducted. The populations  were recLIPC 38 intronic SNPs available to the research community through the KARE project from KoGES were analyzed. The study protocol was approved by the Institutional Review Board of KNIH. The genotyping of the samples from the Ansung and Ansan cohorts was performed using the Affymetrix Genome-Wide Human SNP Array 5.0 . The Bayesian Robust Linear Model with the Mahalanobis distance genotype-calling algorithm was used with the Affymetrix SNP array 5.0. The SNPs were filtered if any of the following criteria were met: i) a call rate lower than 95%, ii) a minor allele frequency (MAF) lower than 0.05, or iii) a significant deviation from the Hardy-Weinberg equilibrium (HWE) lower than 1 × 10-6.All Biochemical data from the KoGES were obtained through the KARE project. Blood samples were collected from the participants after at least eight hours of overnight fasting. HDL-C concentrations were measured with the Advia 1650 analyzer  for the Ansung and Ansan cohorts.LIPC SNPs or diplotypes and HDL-C levels, linear regression analyses were conducted with adjustments for area, age, gender and body mass index (BMI). An additive model was assumed for this study. Log transformation was applied to HDL-C values to normalize their distribution. Associations were evaluated as significant at a level of 0.05 after Bonferroni correction for multiple testing. This conservative (Bonferroni) adjustment required P values of 2.23 × 10-4 in HDL-C before correction, since testing of 224 independent hypotheses (38 single-locus and 186 diplotype tests) for HDL-C was assumed. To determine the effects of SNPs and diplotypes, untransformed HDL-C concentrations were used. Statistical analyses were performed using PLINK version 1.07 (http://pngu.mgh.harvard.edu/~purcell/plink) and Python .To examine possible associations between LIPC 38 intronic SNPs available to the research community through the KARE project, only three SNPs were selected, since these were the only ones with strong associations with HDL-C that were able to withstand Bonferroni correction , on the basis of their diplotypes as well as their SNP genotypes. The SNP rs261332 had the strongest association  with HDL-C levels. Figure The total number of tests of association in the additive model were 224 for HDL-C (data not shown). As shown in Table P value, are shown. The abbreviation b1 means block 1, H12 means the twelfth haplotype cluster, and OH12(*) represents all other haplotypes (including haplotypes below 1% frequency), except the H12 haplotype cluster; b1_H12 is a minor haplotype cluster and b1_OH12(*) a major haplotype cluster. The number of one homozygous diplotype (a b1_H12 pair) was 612, the number of a heterozygous diplotype (b1_H12 and b1_OH12(*)) was 3,064, and the number of another homozygous diplotype (a b1_OH12(*) pair) was 3,858. The strongest association with HDL-C levels showed a P value of 9.09 × 10-19 and was much stronger than the P value of the single SNP rs261332. Figure AAC / AAT or GAC (different in bold) with HDL-C levels was statistically significant, but the association of AAC/AGC (different in bold) with HDL-C levels was not statistically significant in (Table Table 2 = 0.97) [2 = 0.92). The rs2070895 SNP showed a highly significant association, with a 0.057 mmol/l increase per A allele (P = 8 × 10-10) [P = 3.03 × 10-12), with a 0.035 mmol/l increase per T allele. The rs11631342 also showed a significant association (P = 5.66 × 10-6), with a 0.037 mmol/l increase per G allele. The diplotypes b1_H12(AAT-or-GAC)/b1_OH12(*) (Table P = 9.09 × 10-19), with a 0.04 mmol/l increase per (AAT-or-GAC) haplotype cluster. Transporting cholesterol from peripheral tissues to the liver, HL interferes with the interconversions of the mature HDL particles and acts on the triglyceride-rich HDL, which is able to undergo hydrolysis to form small HDL particles, which are then transported to begin anew the process of cholesterol uptake [HL plays a key role in the interconversion between two HDL subspecies and the indirect pathway of hepatic cholesterol uptake in reverse cholesterol transport with involvement of HDL ,14. HDL- = 0.97) , and bot× 10-10) . In the l uptake -20. ThisSignificant haplotype structure has been routinely investigated to identify haplotypes carrying causative mutations. Many software tools like Haploview  and PLINLIPC SNPs or diplotypes and HDL-C levels. The result showed that our approach can identify more precise and distinct signals compared with single-locus tests.We developed a novel method that can yield a synergistic effect by combining the positive aspects of analyses based on haplotype clusters and diplotypes. The Diplotyper software employs Haploview tool, utilizing LD block and haplotypes with a frequency threshold. The software also uses PLINK tool to generate all possible haplotype pairs in given genotypes of samples and perform association analysis using linear or logistic regression. In addition to employing these existing software tools, we designed a new procedure to cluster haplotypes of interest from a large set of haplotypes and implemented a function to generate the patterns of all possible diplotypes from these haplotype clusters. Finally, a function to transform diplotypes into PLINK formats was implemented. All of these processes were fully automated. We tested our method by conducting an association analysis between HDL: high-density lipoprotein; HDL-C: high-density lipoprotein cholesterol; LD: linkage disequilibrium; EM: Expectation-Maximization; SNP: single nucleotide polymorphism; HLA: human leukocyte antigen; ART: assisted reproduction treatments; DTR: diplotype trend regression; HL: hepatic lipase; CEU: Caucasians of European descent from Utah; KARE: Korea Association Resource; KoGES: Korean Genome Epidemiology Study; KNIH: Korean National Institute of Health; MAF: minor allele frequency; HWE: Hardy-Weinberg equilibrium; BMI: body mass index.The authors declare that they have no competing interests.SK designed the algorithm and implemented it for the Diplotyper, and drafted the manuscript. KK conceived of the haplotype cluster-based analysis and helped to draft the manuscript. IK carried out the statistical and physiological analyses. KP and JK participated in the design of the study and the statistical analysis. CS and NC performed data acquisition.This study was supported by a grant from the Korea Health technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A110749), grants from Korea Centers for Disease Control and Prevention , and the Priority Centers Program of the National Research Foundation of Korea (NRF), which is funded by the Ministry of Education, Science, and Technology (No. 2009-0093821).The publication costs for this article were funded by the first author.BMC Medical Genomics Volume 6 Supplement 2, 2013: Selected articles from the Second Annual Translational Bioinformatics Conference (TBC 2012). The full contents of the supplement are available online at http://www.biomedcentral.com/bmcmedgenomics/supplements/6/S2.This article has been published as part of